histoarchitecture and enzyme profile study in interstitial ... bisen and sharad bisen.p… ·...

Int.J.Curr.Microbiol.App.Sci (2019) 8(10): 324-334

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Original Research Article https://doi.org/10.20546/ijcmas.2019.810.033

Histoarchitecture and Enzyme Profile Study in Interstitial Glands

of Non Pregnant, Pregnant and Lactating Indian Leaf-Nosed

Bat Hipposideros speoris (Schneider)

Dharna Bisen1* and Sharad Bisen

2

Department of Entomology, Department of Horticulture, College of Agriculture,

Balaghat, JNKVV, Jabalpur, India

*Corresponding author

A B S T R A C T

Introduction

In the order chiroptera, the patterns and

strategies reproductive are diverse and consist

of the most varied among mammals Peracchi

et al., (2006). Four types of reproductive

patterns are described for bats seasonal

monoestry, seasonal polyestry, non-seasonal

polyestry and bimodal seasonal polyestry

(Fleming et al., 1972, Zortea 2003). The

micro-chiropteran bat Hipposideros speoris

was monoestrun bat and that feed on fruits and

instect. Ovaries of placental mammals exhibit

diversity of structure and function, although

the pivotal role of producing ova and steroid

hormones is common to all species

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 10 (2019) Journal homepage: http://www.ijcmas.com

This study aimed to investigate the relation between interstitial glands, epithelial cords

and pregnancy. The morphological and endocrine aspects of the ovarian interstitial

glands of adult female bats were investigated to establish the probable function and

the biological significance of this compartment in bats. Pregnant and non-pregnant

adult female bats were used for the study. The females were classified according to

their reproductive stages in inactive, active, pregnant, and post-lactating. The

histological and histochemical features of the interstitial glands were studied.

Interstitial glands showed variation in number and morphology during different stages

of pregnancy. The cholesterol and its esters were present in non-pregnant females and

were scarce in pregnant animals. Histochemical study of enzyme like glucose-6-

phosphate dehydrogenase (G-6-PDH), 3β-hydroxesteroid dehydrogenase (3β-HSDH),

succinate dehydrogenase (SDH) and lipids showed variation at different stages of

pregnancy. Our results suggest that the interstitial glands may be storage of precursor

substances for the steroidogenesis. These precursors are probably used when the

endocrine requirements are high, that is during the pregnancy. Thus this compartment

may contribute to the normal gestation of bats. However, the relation between the

interstitial cells epithelial cords and the pregnancy is complex, and further studies are

needed to clearly establish it.

K e y w o r d s

Interstitial glands,

Ovary, Pregnancy

Accepted:

04 September 2019

Available Online:

10 October 2019

Article Info

https://doi.org/10.20546/ijcmas.2019.810.033


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chiropterans display variation of ovarian

structure and functional adaptations that have

few parallels. The interstitial glands of the

ovary are not so well defined as those in the

testis. Hipposideros speoris (order: chiroptera,

suborder Microchiroptera and the family

Rhinolophidae) is a seasonal breeder,

monoesterous and monovular and show

sinistral dominance. The mammalian ovary is

a complex dynamic structure depicting

conspicuous morphological, histochemical,

biochemical and molecular changes involving

follicles corpora lutea and interstitial gland

cells during various reproductive states (Sastry

and Pillai 2005, Dorlikar et al., 2013 and

Godoy et al., 2014). Of the four major

steroidogenically important component of the

mammalian ovary viz-developing follicles,

atretic follicles, corpora lutea and interstitial

gland tissue (Igt), Igt are the most

controversial and neglected component of the

ovary and least studied. Three types of

interstitial gland cells were observed in the

ovaries of the bat Hipposideros speoris thecal,

stromal and epithelial cords all of which

exhibit cyclical variation histologically and

histochemically in relation with reproductive

cycle (Parrott and Skinner 2000, Sastry et al.,

2005, 2008, 2010; Singh et al., 2005, Gill et

al., 2007 and Trivedi and Lall 2007). We

choose to study histochemical localization of

SDH, G-6-PDH, 3β-HSDH and lipids to

examine the site and changing pattern of its

activity in ovaries of Hipposideros speoris

during non-pregnant and pregnant states. The

purpose of the present study provides a brief

account on interstitial gland cells and its role

in steroidogenesis during different stages of

reproduction.

Materials and Methods

Collection of animals and histology :- All

experiments were conducted in accordance

with the principles and procedures approved

by the Departmental research committee,

RTM University Nagpur, Maharastra, India.

More than 20 specimens of Hipposideros

speoris were collected once in a calendar

month with the help of mist net from natural

population inhabiting abandoned mines in

Khapa (2092”N, 7895”E) Nagpur,

Maharashtra throughout the reproductive

cycle. These species are found to inhabit in

cold and humid places preferably dark. All the

bats from the same colony do not show same

pattern of reproductive behavior because there

exists an asynchrony in reproductive cycle

among different females of the same colony.

For histological studies the ovaries were fixed

quickly in Bouin’s fixative, dehydrated in

ethanol and embedded in paraffin wax. The

sections were cut at 5µm, stained with

haematoxylin and eosin.

Histochemical detection of lipids

Ovaries fixed in formol–calcium were cut on

freezing microtome at - 20C and were stained

by Chiffle and Putt method (Lillie and

Fullmer, 1976). These cryocut sections

(10µm) thick were washed briefly in water

and stained with Sudan black B for general

lipids. Lipids appeared black or bluish black.

Histochemical detection of enzymes

For histochemical localization of

hydroxysteroid dehydrogenase (3β); glucose-

6-phophatase dehydrogenase (G-6-PDH) and

succinic dehydrogenase (SDH) sucrose fixed

tissues were cut on cryostat (-20C) at 10µm

thicknesses. The incubation medium of 3β-

HSD and G-6-PDH consisted of nitroblue

tetrazolium (nitro BT), Nicotinamide adenine

dinucleotide (NAD+) and pregnenolone

dissolved in 2-2 dimethylformamide for 3β-

HSD. For G-6-PD the substrate was glucose-

6-phosphate dissolved in 2-2

dimethylformamide. The sections were

incubated in substrate media (di-sodium

succinate) for SDH. Enzyme product was


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visualized by conversion of nitro BT

(nitroblue tetrazolium) to tetrazolium

granules. Appropriate controls were run in

substrate deficient media. The reaction

product colour intensity was visually scored as

+++= strong, ++= moderate, += low, - =

negligible, -- = no reaction (Table-1).

Results and Discussion

The appearance of thecal interstitial gland

tissue observed in this study during pregnant

and non-pregnant phases as described in other

bats, Rhinopoma microphyllum kinneari

(Trivedi and Lall, 2004; 2007); however,

without specifying the reproductive status of

the ovary, origin, development and functional

status notable to clarify the role of these

glands (Singh et al., 2005). The thecal type

interstitial cells originate due to hypertrophy

or transformation of theca interna of atretic

vesicular and multilaminar follicles in

Hipposideros speoris (Fig. 4 and 10). The

thecal Igc are of very transient nature in

Hipposideros speoris (Fig. 1, 7 and 13) as in

few other cases such as women, rhesus

monkey, cow and buffalo as they quickly

revert to the embryonic stromal. Besides

reverting back to the original stromal tissue,

some interstitial tissue in Hipposideros speoris

were also observed to undergo degeneration.

The presence of varying amounts of diffuse

lipids and lipid droplets in the interstitial

tissue signifies its importance as steroidogenic

tissue, as lipid serves as the potential precursor

material for steroid biosynthesis (Fig. 16, 19

and 22).

The thecal interstitial glands of Hipposideros

speoris exhibits a positive reactivity for

steroidogenic enzymes 3β-HSDH (Fig. 16, 19

and 22), SDH (Fig. 34, 37 and 40) and G-6-

PDH (Fig. 43, 46 and 49) during inactive and

active. Such enzyme activities, indicative of

steroid synthesis have been demonstrated in

the interstitial gland cells of thecal origin of

some bats (Singh and Krishna, 1994; Singh et

al., 2005 Trivedi and Lall, 2004, 2007). The

foregoing observations conclude that the

process of atresia in the antral and secondary

follicles (Fig. 1) in Hipposideros speoris

appears to be related to the formation of thecal

type interstitial gland cells, which maintains

ovarian integrity via bio-synthesis, growth rate

of follicle, selection of dominant follicle

which happened to be one in Hipposideros

speoris, as the bat is monovular and mono-

eastrus (Novaldo et al., 2018, Young and

McNeilly 2018).

Both morphological and histochemical studies

reveal that stromal Igc in H. speoris are better

equipped for steroidogenesis (Fig. 2, 5 and

11). The histological changes seem to be

associated with the histochemical activities of

the stromal interstitial cells (Parrott and

Skinner 2000). The intensity of the enzymes

3β-HSD (Fig. 32, 35, 38, 41), G-6-PD (Fig.

55, 56 and 57), SDH (Fig. 44, 47, 50 and 52)

and lipid (Fig. 20, 23, 26 and 29)

accumulation in stromal interstitial cells in the

ovary of Hipposideros speoris showed

variations during the active, pregnant and

lactational phases. Increased lipid

accumulation during inactive phase could be

due to the sharp decline in androgen

production.

All these observations also strongly support

the suggestion that the lipid droplets in the Igc

are the stores of potential precursor materials

which are converted into steroid hormones

when the proper gonadotrophic stimulation

becomes available. So far, the ovarian stroma

(or Igc) has been believed to form mainly

androgens (Guraya, 2000) estrogens (Singh

and Krishna, 1994; Singh et al., 2005) and

progestins (Trivedi and Lall, 2007 and Godoy

et al., 2014). However, in the present work the

observations on the annual reproductive cycle

of the bat, H. speoris emphasizes the synthesis

of androgens (Fig. 1–50).


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The present study also emphasizes that cyclic

changes in the interstitial epithelial cords (EC)

are related to the reproductive cycle of

Hipposiders speoris. No one can noticed this

type of interstitial gland cells earlier in bats

(Gill 2007 and Trivedi and lall 2007). We

observed these Igc cells first in Hipposiders

speoris.

During mid-pregnancy, they were observed to

be highly hypertrophied, closely clustered in

the cortical portion of the ovary (Fig. 9), each

with 20-30 hypertrophied cells in each cord.

Early inactive phase the chords are small and

inactive and there was a sudden burst in SDH,

3β-HSD and G-6-PDH staining profile.

Through the activity of SDH is used as a

criterion of luteal function, its activity is

correlated with presumed sites of hormone

production and places of cellular proliferation

(Sastry and Tembhare 2008, 2009, Sastry et

al., 2010) However G-6-PDH activity was at

more elevated levels during advanced

pregnancy as evident from the occurrence of

density populated, highly hypertrophied zones

in the cortex (Gill et al., 2007, Dorlikar et al.,

2013, Zahra and Farangis 2015, Nivaldo et al.,

2018, Sastry and Pillai 2005, 2008).

Thus we suggest that this compartment may

be a storage precursors for the steroidogenesis

and then, the precursors are principally used

during pregnancy by other ovarian

compartments (e.g.corpus luteum) when the

endocrine requirements are higher.

The observed histological and histochemical

feature of Igc suggest steroidogenic activity,

thus these cells may contribute to the total

endocrine production synthesized by the

ovary. Then the Igc of Hipposiders speoris

may play a role as a source of steroid

precursors during pregnancy and probable

further in its maintenance. However, further

studies are necessary to conform their roles.

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How to cite this article:

Dharna Bisen and Sharad Bisen. 2019. Histoarchitecture and Enzyme Profile Study in

Interstitial Glands of Non Pregnant, Pregnant and Lactating Indian Leaf-Nosed Bat

Hipposideros speoris (Schneider). Int.J.Curr.Microbiol.App.Sci. 8(10): 324-334.

doi: https://doi.org/10.20546/ijcmas.2019.810.033

https://doi.org/10.20546/ijcmas.2019.810.033

histoarchitecture and enzyme profile study in interstitial ... bisen and sharad bisen.p… ·...

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